Enzyme Activities in Rabbit, Guinea Pig, Rat and Mouse Blood Cells after Separation on a Discontinuous Percoli Gradient

Abstract

A technique is described which allows a fractionation of rabbit, guinea pig, rat and mouse blood cells using a discontinuous Percoli gradient. With only two steps of centrifugation a simultaneous isolation of thrombocytes, mononuclear cells, polymorphonuclear cells, and erythrocytes in nearly pure form is performed in a very short time, starting from only 1 ml of blood. In these morphologically almost homogeneous cell fractions the activities of 12 enzymes were determined. The enzyme pattern, which with respect to the number of enzymes, specific cell populations and species was not yet investigated to such an extent, revealed a manifold higher enzyme content in the few cases, in which comparative studies were made. This can be attributed mainly to the completeness of the cell disruption technique using a detergent and certainly also to the use of Percoli as a preferable gradient material.

Catalytic Enzyme Activity Concentration in Plasma of Man, Sheep, Dog, Cat, Rabbit, Guinea Pig, Rat and Mouse. Approach to a Quantitative Diagnostic Enzymology. I. Communication

“…They also were used for the investigation of catalytic activities in various blood cells (12)(13)(14), and other parenchymal tissues (15). Blood from rabbits, dogs and sheep was obtained from animals which were held at the local animal facility for various experimental purposes.…”

“…Considering further the up to 60-fold higher catalytic activities of creatine kinase in platelets of small laboratory animals and dogs compared with man (12,13), it is highly probable that the high values for creatine kinase in serum of these animals originate from platelets during clotting (61,64,65). A plateletderived acid phosphatase is seen in serum but not in plasma (66).…”

The Decline of Catalytic Enzyme Activity Concentration of In Vivo Ageing Erythrocytes of the Man, the Dog and the Rat. Approach to a Quantitative Diagnostic Enzymology, IV. Communication

“…The advantageous properties of Percoll, such äs its low viscosity, low osmotic pressure, non-toxicity and its inability to penetrate cell membranes have resulted in its wide replacement of formerly used gradient material for separating various cell types (8,10,11,14). The employment of a discontinuous Percoll gradient for red cell fractionation with the closest approximation to physiological conditions by using buffered Percoll containing physiological levels of sodium, potassium and albumin was first introduced by Rennie et al (6).…”

“…Cell lysis was performed by the addition of detergent to ensure distribution of nearly all catalytic activity of membrane-integrated or membrane-associated enzyme in the soluble fraction (10,11).…”

“…The least dense Percoll solution with lowest density was used for capturing the occasional (about 2% 0 ) contamination with polymorphonuclear granulocytes. Considering the up to 400-fold higher catalytic activities of certain enzymes in polymorphonuclear cells (e. g. lactate dehydrogenase and phosphohexose isomerase) äs compared to erythrocytes (10,11), inciusion of this first layer could lead to erroneous results. Also both the uppermost and the lowest erythrocyte layers comprise fractions which Stretch theoretically to infinity in each direction and are therefore not clear-cut and well defined populations.…”